Stiffness Tomography by Atomic Force Microscopy Charles Roduit, Serguei Sekatski, Giovanni Dietler, Stefan Catsicas, Frank Lafont, Sandor Kasas  Biophysical Journal  Volume 97, Issue 2, Pages 674-677 (July 2009) DOI: 10.1016/j.bpj.2009.05.010 Copyright © 2009 Biophysical Society Terms and Conditions

Figure 1 FD curves are recorded by indenting (pushing) the tip of the AFM into the sample (a) and by plotting the deformation of the cantilever as a function of the height (b). A hard sample produces a steeper curve (b, dashed red line) than a softer one (b, dashed blue line). In the case where the sample contains harder inclusions (c, red rectangle) located at the L1 level, the FD curve follows the same path as in a (d, green line) initially, but starts to adopt a steeper path just before reaching the L1 level (d, red line). The dashed green line represents the path the curve should follow in the absence of inclusions. By dividing the curve in small segments and by analyzing their individual slope one can detect the presence of the inclusion (d, red and green horizontal bar). Biophysical Journal 2009 97, 674-677DOI: (10.1016/j.bpj.2009.05.010) Copyright © 2009 Biophysical Society Terms and Conditions

Figure 2 Simulation of the indentation process by using the finite elements method. The sample contains inclusions (a) colored in red that have a Young's modulus tree times higher than the bulk of the sample colored in blue. The AFM tip and the spots where indentation was simulated are also represented in blue. During the indentation process the sample deforms as depicted in b. The displacement magnitude is displayed in false colors according to the color bar. (c) The stiffness tomography analysis results. The false colors represent the stiffness in arbitrary units according to the color bar. (d and f) Shows similar simulation using three times stiffer and three times softer platforms with their resulting stiffness tomography in e and g, respectively. The same data scale is used between e and g. One can notice that the stiffness difference appear less contrasted in the case of soft platforms. Arrows in g points to stiffness differences induced by the presence of soft platforms. Biophysical Journal 2009 97, 674-677DOI: (10.1016/j.bpj.2009.05.010) Copyright © 2009 Biophysical Society Terms and Conditions

Figure 3 (a–d) Stiffness tomography images obtained on living neurons. The stiffness, calculated according to the Hertz model, is coded in false colors. Due to the lack of a more accurate model, only color differences are relevant. One can notice the presence of “red” harder inclusions underneath the membrane. The yellow color of the surface of the cells is arbitrary and do not correspond to any stiffness value. The graphs e and f represent the average stiffness of fibroblast as a function of the depth underneath the cell membrane. (e, black curve) Stiffness before the injection of cytochalasin. (e, red curve) Corresponds to the stiffness after the injection. One can notice that in average the cortical part of the cell located under 180 nm became softer after the cytochalasin injection (p < 0.05, two-tailed t-test). (f) The same experiment carried out by injecting the imaging buffer instead of cytochalasin. Biophysical Journal 2009 97, 674-677DOI: (10.1016/j.bpj.2009.05.010) Copyright © 2009 Biophysical Society Terms and Conditions